The sleep/wake cycle is regulated by circadian control of sleep timing and homeostatic control of sleep need. Whereas many details of circadian clock function are known, the molecular and anatomical basis of sleep homeostasis is a mystery. In this proposal, the overall research goal is to use a novel, high-throughput assay involving thermogenetic manipulation of specific arousal circuits in the fly brain to interrogate mechanisms underlying sleep homeostasis. This assay involves transient activation of temperature-sensitive TrpA1 channels in select arousal-controlling neurons in the fly brain. In these animals a mild heat pulse at night stimulates behavioral arousal, thus depriving animals of sleep, which causes subsequent homeostatic recovery sleep the next day. My lab has found that only a subset of wake-promoting neurons is able to elicit sleep homeostasis, indicating that this subset likely falls within a homeostatic circuit, and this work has led to the development of sleep homeostasis circuit model in which the activity of sleep-promoting (S) neurons is upregulated following sustained activity of wake-promoting (W) neurons. Consistent with the existence of S neurons, I have found that electrically silencing specific neurons with transgenic potassium channels suppresses sleep homeostasis without obviously affecting W neuron function or daily fluctuations in the sleep/wake cycle. Goals of this proposal are to describe the roles of molecules and changes in neuronal activity that contribute to sleep homeostasis and to determine the contribution of sleep homeostatic molecules and circuitry to learning and memory. The experiments in aim 1 focus on localization and functional relevance of two genes, identified in a thermogenetic screen, that are required for sleep homeostasis. I will use RNAi knockdown against these genes in different areas of the brain to refine where their expression is required for functional sleep homeostasis. In Aim 2 I will identify additional components of sleep homeostasis circuitry by screening for neurons which, when electrically silenced, selectively suppress homeostatic recovery sleep. I will also express different genetically encoded fluorophores in distinct identified components of sleep homeostasis circuitry to determine their anatomical relationships. In Aim 3 I will address the role of sleep homeostasis in short-term learning and memory using a robust aversive-taste training assay. Understanding the mechanisms that regulate sleep need may improve our understanding of homeostatically controlled processes in the nervous system and open the door to pharmacological intervention in sleep disorders that currently detract from human health and well-being.